The microRNA-27a: ZBTB10-specificity protein pathway is involved in follicle stimulating hormone-induced VEGF, Cox2 and survivin expression in ovarian epithelial cancer cells.

Previously, we demonstrated that follicle stimulating hormone (FSH) enhanced VEGF expression and facilitated ovarian cancer angiogenesis via the PI3K/AKT signaling pathway. In this study, we further investigated the involvement of microRNA-27a: ZBTB10­specificity protein pathway in the mechanism of FSH-induced VEGF, Cox2 and survivin expression. Treatment with FSH resulted in significant increase in the expression of VEGF, Cox2, survivin, Sp1 proteins and microRNA-27a in a dose-dependent manner, whereas reverse protein expression pattern was observed in ZBTB10. Downregulation of microRNA-27a using antisense microRNA-27a blocked FSH-induced VEGF, Cox2 and survivin expression. Overexpression of ZBTB10 also attenuated the FSH-induced expression of these molecules. The enhanced expression of VEGF, Cox2 and survivin was also abolished by knocking down Sp1 using small interfering RNA. Collectively, these results indicated that stimulation of ovarian cancer cell VEGF, Cox2 and survivin expression by FSH involves the microRNA­27a: ZBTB10-specificity protein pathway.

Structural and functional insights into lipid-bound nerve growth factors.

Nerve growth factor (NGF) is a dimeric molecule that modulates the survival, proliferation, and differentiation of nervous cells and is also known to act on cells of the immune system and endocrine system. NGFs extracted from mouse submaxillary gland and cobra venom have different immunological behaviors, yet the underlying mechanism remains unclear. Here we report the crystal structure of the NGF purified from Chinese cobra Naja naja atra (cNGF), which unexpectedly reveals a 2-tailed lipid molecule that is embedded between the two protomers of the NGF homodimer. In addition, crystallographic analysis indicated that the purified mouse NGF(mNGF) is free from lipid but can bind lysophosphatidylserine (lyso-PS) in the same pocket as cNGF. Bioassays indicated that the binding of lipid molecules to cNGF and mNGF are essential for their mast cell activation activity and abates their p75(NTR) binding capacity. Taken together, these results suggest a new mechanism for the regulation of the function of NGF.

Structural and functional characterization of ryanodine receptor-natrin toxin interaction.

Cysteine-rich secretory proteins (CRISPs) are widely distributed, and notably occur in the mammalian reproductive tract and in the salivary glands of venomous reptiles. Most CRISPs can inhibit ion channels, such as the cyclic nucleotide-gated ion channel, potassium channel, and calcium channel. Natrin is a CRISP that has been purified from snake venom. Its targets include the calcium-activated potassium channel, the voltage-gated potassium channel, and the calcium release channel/ryanodine receptor (RyR). Immunoprecipitation experiments showed that natrin binds specifically to type 1 RyR (RyR1) from skeletal muscle. Natrin was found to inhibit both the binding of ryanodine to RyR1, and the calcium-channel activity of RyR1. Cryo-electron microscopy and single-particle image reconstruction analysis revealed that natrin binds to the clamp domains of RyR1. Docking of the crystal structure of natrin into our cryo-electron microscopy density map of the RyR1 + natrin complex suggests that natrin inhibits RyR1 by stabilizing a domain-domain interaction, and that the cysteine-rich domain of natrin is crucial for binding. These findings help reveal how natrin toxin inhibits the RyR calcium release channel, and they allow us to posit a generalized mechanism that governs the interaction between CRISPs and ion channels.

Structural and functional analysis of natrin, a venom protein that targets various ion channels.

Cysteine-rich secretory proteins (CRISPs) are secreted single-chain proteins found in different sources. Natrin is a member of the CRISP family purified from the snake venom of Naja naja atra, which has been reported as a BKca channel blocker. In our study, crystals of natrin were obtained in two different crystal forms and the structure of one of them was solved at a resolution of 1.68A. Our electrophysiological experiments indicated that natrin can block the ion channel currents of the voltage-gated potassium channel Kv1.3. Docking analyses of the interaction between natrin and Kv1.3 revealed a novel interaction pattern different from the two previously reported K(+) channel inhibition models termed "functional dyad" and "basic ring". These findings offered new insights into the function of natrin and how the specific interactions between CRISPs and different ion channels can be achieved.

Structure of a king cobra phospholipase A2 determined from a hemihedrally twinned crystal.

An acidic PLA(2) (OH APLA(2)-II) from the venom of Ophiophagus hannah (king cobra) shows greater phospholipase A(2) activity and weaker cardiotoxic and myotoxic activity than a homologous acidic PLA(2) from the same venom. The crystal of the enzyme belongs to space group P6(3). The crystals are invariably hemihedrally twinned, exhibiting perfect 622 Laue symmetry. The structure was determined by molecular replacement and refined using a hemihedral twinning program at 2.1 A resolution. The final model has reasonable stereochemistry and a crystallographic R factor of 19.5% (R(free) = 21.5%). The structure reveals the molecular arrangement and the mode of twinning. There are six independent molecules in the asymmetric unit. Owing to the presence of a non-crystallographic twofold parallel to the hemihedral twinning twofold, the molecular packing in the twinned crystal is extremely similar to that in an untwinned crystal for four of the molecules. This unique molecular arrangement may be related to the difficulty in recognizing the twinning. The structure was compared with the previously determined structure of a homologous acidic PLA(2) from the same source. The comparison shows structural changes that might be implicated in the increased catalytic activity and weakened toxicity.

Crystallization and preliminary X-ray diffraction studies of cobra venom beta-nerve growth factor.

beta-Nerve growth factor (beta-NGF) is a 118 amino acid residue polypeptide with an important role in the survival and development of certain neuronal populations. A beta-NGF purified from cobra venom was crystallized by the hanging-drop vapor diffusion method. The crystals belong to the tetragonal space group P4(1)2(1)2 (or its enantiomorph P4(3)2(1)2) with unit-cell parameters a=b=61.75, c=154.40A. X-ray data from these crystals were collected to 3.2A resolution. Analysis of the packing density shows that the asymmetric unit probably contains two molecules. The self-rotation calculation implied that a beta-NGF dimer might exist in the asymmetric unit.

Structure of a cardiotoxic phospholipase A(2) from Ophiophagus hannah with the "pancreatic loop".

The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

Preliminary crystallographic study of an acidic phospholipase A2 from Ophiophagus hannah (king cobra).

An acidic phospholipase A(2) (OH APLA(2)-II) with an isoelectric point (pI) of 4.0 was recently isolated from Ophiophagus hannah (king cobra) from Guangxi province, China. Comparison of this enzyme to a previously reported homologous phospholipase A(2) from the same venom shows that it lacks toxicity and exhibits a greater phospholipase activity. OH APLA(2)-II has been crystallized by the hanging-drop vapour-diffusion method using 1,6-hexanediol and magnesium chloride as precipitants. The crystal belongs to space group P6(3), with unit-cell parameters a = b = 98.06, c = 132.39 A. The diffraction data were collected under cryoconditions (100 K) and reduced to 2.1 A resolution. A molecular-replacement solution has been determined and shows that there are six molecules in one asymmetric unit.

Crystal structures of an acidic phospholipase A(2) from the venom of Naja Kaouthia.

A phospholipase A(2) purified from the venom of Naja kaouthia (Guangxi cobra) exhibits anticoagulant activities. The structures of two crystal forms were determined by X-ray crystallography at 2.8A resolution with the Naja naja (India cobra) PLA(2) as an initial model. The enzyme exhibits a trimer structure, which is similar to that of India cobra PLA(2). This reinforces the physiological relevance of the oligomer. The trimer has a wide cavity, which allows the substrate to enter and interact with the catalytic site. The formation of the trimer may serve as a storage method to improve the solubility at high concentration in the venom gland. The Ca2+ binding loop in the absence of the cation can exist in different conformations depending on its surroundings.

Preliminary Crystallographic Analysis of Phospholipase A(2) from Naja naja kaouthia Lesson Venom.

An acidic phospholipase A(2) isolated from the venom of Naja naja kaouthia Lesson in Guangxi exhibits anticoagulative and hemolytic activities. In this work, the enzyme was crystallized by the method of hanging drop vapor diffusion. Two crystal forms were obtained and characterized by X-ray diffraction. One of them belonged to space group P 4 ( 3 ) 2 ( 1 )2 or P4(1)2(1)2 with unit cell parameters a b 8.797 nm, c 10.831 nm and there were three molecules per asymmetric unit the other belonged to space group P2(1)3 with unit cell parameters a b c 6.840 nm and there was one molecule per asymmetric unit. The diffraction data were collected up to 0.28 nm for each crystal form. The crystal properties of Naja naja verom phospholipase A(2) from different geographical regions are compared.

Cloning and Sequence Analysis of cDNAs Encoding Two Acidic PLA(2) from venom of Ophiophagus hannah(King Cobra), Guangxi Species.

Total RNA was extracted from venom glands of Ophiophagus hannah, Guangxi species. The cDNAs encoding PLA(2) were amplified by RT-PCR and cloned into the PUCm-T vector. The positive clones encoding two acidic PLA(2) (APLA(2)-1 and APLA(2)-2) were selected and bidirectionally sequenced. Their complete amino acid sequences were deduced and found to be identical to the known amino acid sequences. Their isoelectric points calculated by computer agreed with the values determined with their protein. Homology analysis indicated that the mature peptide of APLA(2)-1 had high homology with PLA(2) from venoms of Ophiophagus hannah, Fujian and Taiwan species, but APLA(2)-2 had lower homology. The most striking difference between APLA(2)-2 and other PLA(2) from Ophiophagus hannah venoms is the missing of a extra "pancreatic loop" at residues 62--66 in APLA(2)-2, and it may be related to their species evolution and biological activity.

NGF-Tf Conjugate Prevents Degeneration of Substantia Nigra Neurons in a Mouse Model of Parkinson's Disease.

Nerve growth factor(NGF) was purified from Naja naja atra snake venom and conjugated to transferrin(Tf). This conjugate was intravenously injected into a mouse model of Parkinson's disease (PD). Both immunohistochemical staining and pathological detection showed that the NGF-Tf conjugate could prevent the loss of tyrosine hydroxylase-immunoreactive neurons located in substantia nigra and the cell counts of NGF group were 2 330.0+/-260.3, and those of the MPTP group and the control group were 797.0+/-121.4 and 2 381.0+/-158.0, respectively. In addition, electron microscopic examination revealed significant protection against demyelination and vacuolation in subtantia nigra neurons in contrast to the control group. The i.v. injected NGF-Tf conjugate also reversed the neurodegenerative changes such as karyopyknosis, chromatolysis and intracytoplasmic inclusion in diseased neurons.